Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
A major experimental difficulty in unraveling neuronal circuits is the identification of the postsynaptic targets of a given neuron. While ultrastructural reconstructions of target cells are labor intensive, simultaneous recordings from pairs of randomly chosen cells have been recently used to identify connected pairs of neurons. The present study describes a novel approach using fluorescent (e.g. calcium) imaging of populations of neurons that allows us to rapidly identify many postsynaptic targets of any given neuron in a brain slice. Combining bulk-loading of fluorescent (e.g. calcium) indicators with optimal imaging and analysis protocols, the present study demonstrates that neurons that have somatic calcium transients time-locked to the spikes of a stimulated neuron are monosynaptically connected to it. This method can be supplemented with recordings from identified postsynaptic neurons and can be applied systematically to quickly reconstruct circuits from many regions of the CNS.
One of the most neglected aspects of systems neurobiology is the reconstruction of specific circuit diagrams involved in the particular function studied. It is evident that the knowledge of the detailed circuit diagram is necessary to fully understand the computations carried out by any nervous system. Yet, even in cases where the nature of the computation is clear, the detailed implementation of the computational algorithms by circuits of neurons remains mysterious (Heiligenberg, 1991). A direct approach to decipher a particular circuit is its reconstruction with electron microscopy. Such a program has only been successfully achieved in the nervous system of C. elegans, which has only 302 neurons and with a stereotyped connectivity from animal to animal (White et al., 1986). Until the advent of automatic electron microscopy reconstructions (Macagno et al., 1979; White et al., 1993), these efforts were impractical in most nervous systems due to the number of neurons and the laboriousness of the task. More specifically, one of the major practical problems facing the electron microscopists is identifying the neurons targeted by a particular axon from an identified neuron. Only detailed serial thin-section reconstructions of the dendritic tree of the target neuron can reveal, in most instances, its identity (McGuire et al., 1991; Czeiger and White, 1993).
In recent decades, the use of multielectrode electrical recordings has provided an alternative strategy to characterizing circuit connectivity. Extracellular recordings using microelectrode arrays permit the simultaneous recordings of dozen or hundreds of neurons (Meister et al., 1994). While extremely valuable for reconstructing the spatio-temporal dynamics of a neuronal population (Meister et al., 1991), this approach, however, suffers the disadvantage of sampling only a small proportion of the neurons in a given area and also of lacking anatomical information about the particular cells responsible for the spikes. Another approach to the functional circuitry is to perform dual intracellular recordings from pairs of connected neurons. This has been achieved in brain slices by combining intracellular recording and staining with electron microscopy reconstructions and confirmation of the contact (Gulyas et al., 1993; Buhl et al., 1994; Deuchards et al., 1994). In principle, this approach could accurately characterize the functional connectivity between pairs of cortical neurons. Nevertheless, given the difficulty of obtaining recordings of pairs of connected neurons, the many different types of neurons, and the low probability that any given pairxe2x80x94chosen at randomxe2x80x94is actually connected, would make only a labor-intensive effort with semi-automated experimental procedures successful.
The studies herein describe a novel method to identify postsynaptic targets from a given neuron in brain slices stained with fluorescent (e.g. calcium) indicators. This technique builds on previous work that showed that optical monitoring of the activity of a neuronal population is feasible with calcium indicators (Yuste and Katz, 1991; O""Donovan et al., 1993). Improvements in bulk loading methods for calcium indicators (Tsien, 1981; Schwartz et al., 1998; Yuste, 1999) and the ubiquitous presence of calcium channels and their activation by action potentials (Yuste and Denk, 1995; Helmchen et al., 1996) makes possible the optical detection of action potentials in populations of neurons (Smetters et al., 1999) and consequently enables the detection of which neurons produce an action potential in response to stimulation of another neuron.
This invention provides a method of identifying a connection between a first neuron and a second neuron or plurality of neurons comprising of: a) loading neurons in a neural tissue slice with a fluorescent indicator; b) optically detecting an image of the fluorescent indicator-loaded neurons; c) stimulating a first neuron in the neural tissue slice to elicit one action potential or many (multiple) action potentials from said neuron; and d) optically detecting a change in intensity of the fluorescent indicator in the image at a second neuron or plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first neuron in step (c), wherein detection of a transient decrease in the intensity of the fluorescent indicator in the image at the second neuron or plurality of neurons indicates the activation in the second neuron or plurality of neurons, thereby identifying the connection between the second neuron or plurality of neurons and the first neuron.
This invention provides a method of detecting the effect of a neuromodulator on a connection between a first neuron and a second neuron or a plurality of neurons forming a circuit which comprises: a) loading neurons in a neural tissue slice with a fluorescent indicator; b) optically detecting an image of the fluorescent indicator-loaded neurons; c) administering a neuromodulator to the neural tissue slice to modulate the eliciting of one action potential or many (multiple) action potentials from the neurons or the plurality of neurons forming the circuit and to modulate the activity of a synaptic connection or plurality of connections between the neurons; and d) optically detecting a change in intensity of the fluorescent indicator in the image at the second neuron or in the plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first neuron in step (c), wherein detection of a transient decrease or increase in the intensity of the fluorescent indicator in the image at the second neuron or the plurality of neurons indicates an increase or decrease, respectively, in the acivation of the second neuron or plurality of neurons, thereby identifying the effect of the neuromodulator on the connection between the first neuron and the second neuron or between the plurality of neurons forming the circuit.
This invention provides a method of identifying an inhibitory connection between a first neuron and a second neuron or plurality of neurons comprising of: a) loading neurons in a neural tissue slice with a fluorescent indicator; b) optically detecting an image of the fluorescent indicator-loaded neurons under a high background of spontaneous activity; c) stimulating a first inhibitory neuron in the neural tissue slice to elicit one action potential or many (multiple) action potentials from said neuron; and d) optically detecting a change in intensity or lack thereof (no intensity) of the fluorescent indicator in the image at a second neuron or plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first inhibitory neuron in step (c), wherein detection of a transient decrease in the intensity of the fluorescent indicator in the image at the second neuron or plurality of neurons indicates an inactivation of the second neuron or plurality of neurons, thereby identifying the inhibitory connection between the first inhibitory neuron and the second neuron or plurality of neurons.
This invention provides a method of detecting the effect of a neuromodulator on an inhibitory connection between a first neuron and a second neuron or a plurality of neurons forming a circuit which comprises: a) loading neurons in a neural tissue slice with a fluorescent indicator; b) optically detecting an image of the fluorescent indicator-loaded neurons; c) administering a neuromodulator to the neural tissue slice to modulate the eliciting of one action potential or many (multiple) action potentials from the neurons or the plurality of neurons forming the circuit and to modulate the activity of a synaptic connection or plurality of connections between the neurons; and d) optically detecting a change in intensity of the fluorescent indicator in the image at the second neuron or in the plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first neuron in step (c), wherein detection of a transient decrease or increase in the intensity of the fluorescent indicator in the image at the second neuron or the plurality of neurons indicates, indicates an activation or inactivation, respectively, of the second neuron or plurality of neurons, thereby identifying the effect of the neuromodulator on the inhibitory connection between the first neuron and the second neuron or between the plurality of neurons forming the circuit.
This invention provides a method of identifying an inhibitory connection between a first neuron and a second neuron or plurality of neurons comprising of: a) loading neurons in a neural tissue slice with a chloride indicator; b) optically detecting an image of the chloride indicator-loaded neurons; c) stimulating a first inhibitory neuron in the neural tissue slice to elicit one action potential or many action potentials from said neuron; and d) optically detecting a change in intensity of the chloride indicator in the image at a second neuron or plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first inhibitory neuron in step (c), wherein detection of a transient change in the intensity of the chloride indicator in the image at the second neuron or plurality of neurons indicates an inhibitory effect in the second neuron or plurality of neurons, thereby identifying the inhibitory connection between the first inhibitory neuron and the second neuron or plurality of neurons.
This invention provides a method of detecting the effect of a neuromodulator on an inhibitory connection between a first neuron and a second neuron or a plurality of neurons forming a circuit which comprises: a) loading neurons in a neural tissue slice with a chloride indicator; b) optically detecting an image of the chloride indicator-loaded neurons; c) administering a neuromodulator to the neural tissue slice to modulate the eliciting of one action potential or many (multiple) action potentials from the neurons or the plurality of neurons forming the circuit and to modulate the activity of a synaptic connection or plurality of connections between the neurons; and d) optically detecting a change in intensity of the chloride indicator in the image at the second neuron or in the plurality of neurons of the neural tissue slice in response to the action potential(s) elicited from the first neuron in step (c), wherein detection of a transient change in the intensity of the chloride indicator in the image at the second neuron or the plurality of neurons indicates, indicates an inhibitory effect on the second neuron or plurality of neurons, thereby identifying the effect of the neuromodulator on the inhibitory connection between the first neuron and the second neuron or between the plurality of neurons forming the circuit.
This invention provides a method of identifying a connection between a first neuron and a second neuron or plurality of neurons in vivo comprising of: a) genetically encoding a transgenic animal to express GFP based-indicators in neural tissue; b) optically detecting an image of the GFP-based indicator in neurons of a neural tissue of the transgenic mouse using two-proton microscopes; c) stimulating a first neuron or a neural circuit in the neural tissue to elicit multiple action potentials from said neuron or said neural circuit; and d) optically detecting a change in intensity of the GFP-based indicator in the image at a second neuron or plurality of neurons of the neural tissue in response to the multiple action potentials elicited from the first neuron in step (c), wherein detection of a transient change in the intensity of the GFP-based indicator in the image at the second neuron or plurality of neurons indicates activation or inactivation of the second neuron or plurality of neurons, thereby identifying the connection between the second neuron or plurality of neurons and the first neuron.
This invention provides a method of detecting the effect of a neuromodulator on a connection between a first neuron and a second neuron or plurality of neurons in vivo which comprises: a) genetically encoding a transgenic animal to express GFP based-indicators in neural tissue; b) optically detecting an image of the GFP based indicator in neurons of a neural tissue in the transgenic mouse using a conventional microscope or a two-proton microscope; c) administering a neuromodulator to the neural tissue of the transgenic animal to modulate the eliciting of one action potential or many (multiple) action potentials from the neurons or the plurality of neurons forming the circuit and to modulate the activity of a synaptic connection or plurality of connections between the neurons; and d) optically detecting a change in intensity of the GFP-based indicator in the image at a second neuron or plurality of neurons of the neural tissue in response to the action potential(s) elicited from the first neuron in step (c), wherein detection of a transient change in the intensity of the GFP-based indicator in the image at the second neuron or plurality of neurons indicates activation or inactivation (inhibition) of the second neuron or plurality of neurons, thereby identifying the effect of the neuromodulator on the connection between the first neuron and the second neuron or plurality of neurons in vivo.
This invention provides a method of identifying a connection between a first neuron and a second neuron or plurality of neurons in vitro comprising of: a) optically detecting an image of the GFP-based indicator in neurons of a neural tissue slice from a transgenic animal whose neural tissue expresses GFP-based indicators using a conventional microscope or a two-proton microscope; b) stimulating a first neuron or a neural circuit in the neural tissue slice to elicit multiple action potentials from said neuron or said neural circuit; and c) optically detecting a change in intensity of the GFP-based indicator in the image at a second neuron or plurality of neurons of the neural tissue in response to the multiple action potentials elicited from the first neuron in step (b), wherein detection of a transient change in the intensity of the GFP-based indicator in the image at the second neuron or plurality of neurons indicates activation or inactivation (inhibition) of the second neuron or plurality of neurons, thereby identifying the connection between the second neuron or plurality of neurons and the first neuron.
This invention provides a method of detecting the effect of a neuromodulator on a connection between a first neuron and a second neuron or plurality of neurons in vitro which comprises: a) optically detecting an image of the GFP-based indicator in neurons of a neural tissue slice from a transgenic animal whose neural tissue expresses GFP-based indicators using a conventional microscope or a two-proton microscope; b) administering a neuromodulator to the neural tissue of the transgenic animal to modulate the eliciting of one action potential or many (multiple) action potentials from the neurons or the plurality of neurons forming the circuit and to modulate the activity of a synaptic connection or plurality of connections between the neurons; and c) optically detecting a change in intensity of the GFP-based indicator in the image at a second neuron or plurality of neurons of the neural tissue in response to the action potential(s) elicited from the first neuron in step (b), wherein detection of a transient change in the intensity of the GFP-based indicator in the image at the second neuron or plurality of neurons indicates activation or inhibition of the second neuron or plurality of neurons, thereby identifying the the effect of the neuromodulator on the connection between the first neuron and the second neuron or plurality of neurons in vitro.